The present invention relates to immunogenic compositions for protection against disease caused by Chlamydia infection in mammals, including humans.
Chlamydiae are procaryotes. They exhibit morphologic and structural similaries to gram negative bacteria, including a trilaminar outer membrane, which contains lipopolysaccharide and several membrane proteins Chlamydiae are differentiated from other bacteria by their morphology and by a unique developmental cycle. They are obligate intracellular parasites with a unique biphasic life cycle consisting of a metabolically inactive but infectious extracellular stage and a replicating but non-infectious intracellular stage. The replicative stage of the life-cycle takes place within a membrane-bound inclusion which sequesters the bacteria away from the cytoplasm of the infected host cell.
Because chlamydiae are small and multiply only within susceptible cells, they were long thought to be viruses. However, they have many characteristics in common with other bacteria: (1) they contain both DNA and RNA, (2) they divide by binary fission, (3) their cell envelopes resemble those of other gram-negative bacteria, (4) they contain ribosomes similar to those of other bacteria, and (5) they are susceptible to various antibiotics. Chlamydiae can be seen in the light microscope, and the genome is about one-third the size of the Escherichia coli genome.
Many different strains of chlamydiae have been isolated from birds, man and other mammals, and these strains can be distinguished on the basis of host range, virulence, pathogenesis, and antigenic composition. There is strong homology of DNA within each species, but surprisingly little between species, suggesting longstanding evolutionary separation.
C. trachomatis has a high degree of host specificity, being almost completely limited to man, and causes ocular and genitourinary infections of widely varying severity. In contrast, C. psittaci strains are rare in man but are found in a wide range of birds and also in wild, domestic, and laboratory mammals, where they multiply in cells of many organs.
C. pneumoniae is a common human pathogen, originally described as the TWAR strain of C. psittaci, but subsequently recognized to be a new species. C. pneumoniae is antigenically, genetically, and morphologically distinct from other Chlamydia species (C. trachomatis, C. pecorum and C. psittaci). It shows 10% or less DNA sequence homology with either of C. trachomatis or C. psittaci and so far appears to consist of only a single strain, TWAR.
C. pneumoniae is a common cause of community acquired pneumonia, less frequent only than Streptococcus pneumoniae and Mycoplasma pneumoniae (refs. 1 and 2xe2x80x94Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure). C. pneumoniae can also cause upper respiratory tract symptoms and disease, including bronchitis and sinusitis (refs. 1 to 4). The great majority of the adult population (over 60%) has antibodies to C. pneumoniae (ref. 5), indicating past infection which was unrecognized or asymptomatic.
C. pneumoniae infection usually presents as an acute respiratory disease (i.e., cough, sore throat, hoarseness, and fever; abnormal chest sounds on auscultation). For most patients, the cough persists for 2 to 6 weeks, and recovery is slow. In approximately 10% of these cases, upper respiratory tract infection is followed by bronchitis or pneumonia. Furthermore, during a C. pneumoniae epidemic, subsequent co-infection with pneumococcus has been noted in about half of these pneumonia patients, particularly in the infirm and the elderly. As noted above, there is more and more evidence that C. pneumoniae infection is also linked to diseases other than respiratory infections.
The reservoir for the organism is presumably people. In contrast to C. psittaci infections, there is no known bird or animal reservoir. Transmission has not been clearly defined, but may result from direct contact with secretions, from formites, or from airborne spread. There is a long incubation period, which may last for many months. Based on analysis of epidemics, C. pneumoniae appears to spread slowly through a population (case-to-case interval averaging 30 days) because infected persons are inefficient transmitters of the organism. Susceptibility to C. pneumoniae is universal. Reinfections occur during adulthood, following the primary infection as a child. C. pneumoniae appears to be an endemic disease throughout the world, noteworthy for superimposed intervals of increased incidence (epidemics) that persist for 2 to 3 years. C. trachomatis infection does not confer cross-immunity to C. pneumoniae. Infections are easily treated with oral antibiotics, tetracycline or erythromycin (2 g/d, for at least 10 to 14 d). A recently developed drug, azithromycin, is highly effective as a single-dose therapy against chlamydial infections.
In most instances, C. pneumoniae infection is mild and without complications, and up to 90% of infections are subacute or unrecognized. Among children in industrialized countries, infections have been thought to be rare up to the age of 5 years, although a recent study has reported that many children in this age group show PCR evidence of infection despite being seronegative, and estimates a prevalence of 17 to 19% in 2 to 4 years old (ref. 6). In developing countries, the seroprevalence of C. pneumoniae antibodies among young children is elevated, and there are suspicions that C. pneumoniae may be an important cause of acute lower respiratory tract disease and mortality for infants and children in tropical regions of the world.
From seroprevalence studies and studies of local epidemics, the initial C. pneumoniae infection usually happens between the ages of 5 and 20 years. In the USA, for example, there are estimated to be 30,000 cases of childhood pneumonia each year caused by C. pneumoniae. Infections may cluster among groups of children or young adults (e.g., school pupils or military conscripts).
C. pneumoniae causes 10 to 25% of community-acquired lower respiratory tract infections (as reported from Sweden, Italy, Finland, and the USA). During an epidemic, C. pneumonia infection may account for 50 to 60% of the cases of pneumonia. During these periods, also, more episodes of mixed infections with S. pneumoniae have been reported.
Reinfection during adulthood is common; the clinical presentation tends to be milder. Based on population seroprevalence studies, there tends to be increased exposure with age, which is particularly evident among men. Some investigators have speculated that a persistent, asymptomatic C. pneumoniae infection state is common.
In adults of middle age or older, C. pneumoniae infection may progress to chronic bronchitis and sinusitis. A study in the USA revealed that the incidence of pneumonia caused by C. pneumoniae in persons younger than 60 years is 1 case per 1,000 persons per year; but in the elderly, the disease incidence rose three-fold. C. pneumoniae infection rarely leads to hospitalization, except in patients with an underlying illness.
Of considerable importance is the association of atherosclerosis and C. pneumoniae infection. There are several epidemiological studies showing a correlation of previous infections with C. pneumoniae and heart attacks, coronary artery and carotid artery disease (refs. 7 to 11). Moreover, the organisms has been detected in atheromas and fatty streaks of the coronary, carotid, peripheral arteries and aorta (refs. 12 to 16). Viable C. pneumoniae has been recovered from the coronary and carotid artery. (refs, 17, 18). Furthermore, it has been shown that C. pneumoniae can induce changes of atherosclerosis in a rabbit model (ref. 19). Taken together, these results indicate that it is highly probable that C. pneumoniae can cause atherosclerosis in humans, though the epidemiological importance of chlamydial atherosclerosis remains to be demonstrated.
A number of recent studies have also indicated an association between C. pneumoniae infection and asthma. Infection has been linked to wheezing, asthmatic bronchitis, adult-onset asthma and acute exacerbation of asthma in adults, and small-scale studies have shown that prolonged antibiotic treatment was effective at greatly reducing the severity of the disease in some individuals (refs. 20 to 25).
In light of these results, a protective vaccine against disease caused by C. pneumoniae infection would be of considerable importance. There is not yet an effective vaccine for human C. pneumoniae infection. Nevertheless, studies with C. trachomatis and C. psittaci indicate that this is an attainable goal. For example, mice which have recovered from a lung infection with C. trachomatis are protected from infertility induced by a subsequent vaginal challenge (ref. 26). Similarly, sheep immunized with inactivated C. psittaci were protected,from subsequent chlamydial-induced abortions and stillbirths (ref. 27). Protection from chlamydial infections has been associated with Th1 immune responses, particularly the induction of INFxcex3-producing CD4+ T cells (ref. 28). The adoptive transfer of CD4+ cell lines or clones to nude or SCID mice conferred protection from challenge or cleared chronic disease (refs. 29, 30) and in vivo depletion of CD4+ T cells exacerbated disease post-challenge (refs. 31, 32). However, the presence of sufficiently high titres of neutralizing antibody at mucosal surfaces can also exert a protective effect (ref. 33).
The extent of antigenic variation within the species C. pneumoniae is not well characterized. Serovars of C. trachomatis are defined on the basis of antigenic variation in major outer membrane proteins (MOMP), but published C. pneumoniae MOMP gene sequences show no variation between several diverse isolates of the organism (refs. 34, 35, 36). Regions of the protein known to be conserved in other chlamydial MOMPs are conserved in C. pneumoniae (refs. 34, 35). One study has described a strain of C. pneumoniae with a MOMP of greater that usual molecular weight, but the gene for this has not been sequenced (ref. 1). Partial sequences of outer membrane protein 2 from nine diverse isolates were also found to be invariant (ref. 17). The genes for HSP60 and HSP70 show little variation from other chlamydial species, as would be expected. The gene encoding a 76 kDa antigen has been cloned from a single strain of C. pneumoniae. It has no significant similarity with other known chlamydial genes (ref. 4).
Many antigens recognized by immune sera to C. pneumoniae are conserved across all chlamydiae, but 98 kDa, 76 kDa and 54 kDa proteins may be C. pneumoniae specific (refs. 2, 4, 37). Immunoblotting of isolates with sera from patients does show variation of blotting patterns between isolates, indicating that serotypes C. pneumoniae may exist (refs. 1, 17). However, the results are potentially confounded by the infection status of the patients, since immunoblot profiles of a patient""s sera change with time post-infection. An assessment of the number and relative frequency of any serotypes, and the defining antigens, is not yet possible.
Thus, a need remains for effective compositions for preventing and treating Chlamydia infections.
The present invention provides a novel approach to immunizing against Chlamydial infection based on nucleic acid immunization. It has surprisingly been found that the administration of a combination of nucleotide sequences encoding two different chlamydial proteins provides an enhanced protection efficacy.
Accordingly, in one aspect of the present invention, there is provided an immunogenic composition for in vivo administration to a host for the generation in the host of a protective immune response against Chlamydial infection, comprising a first vector comprising a first nucleotide sequence encoding a major outer membrane protein (MOMP) of a strain of Chlamydia and a first promoter sequence operatively coupled to said first nucleotide sequence for expression of said MOMP in the host; a second vector comprising a second nucleotide sequence encoding a 76 kDa protein of a strain of Chlamydia and a second promoter sequence operatively coupled to said second nucleotide sequence for expression of said 76 kDa protein in the host; and a pharmaceutically-acceptable carrier therefor.
The first nucleotide sequence may encode a MOMP from any strain of Chlamydia, preferably from C. pneumoniae but also including C. trachomatis. The second nucleotide sequence encoding the MOMP protein of C. pneumoniae may have SEQ ID No: 12, 13 or 14 or may encode a MOMP having a SEQ ID No: 1.5 or 16.
The first promoter which is employed may be a cytomegalovirus promoter, although an ther convenient promoter may be employed.
The second nucleotide sequence may encode a 76 kDa protein from any strain of Chlamydia, preferably from C. pneumoniae but also including C. trachomatis. The second nucleotide sequence encoding the 76 kDa protein of C. pneumoniae may have SEQ ID No: 1, 2, 3 or 4. The second nucleotide sequence may encode a 76 kDa protein having a molecular size of about 35 kDa and having SEQ ID No: 7 or may encode a 76 kDa protein having a molecular size of about 60 kDa and having SEQ ID No: 8 or 9.
The second promoter which is employed may be a cytomegalovirus promoter, although any other convenient promoter may be employed.
The first vector preferably comprises a plasmid vector and specifically may be pCAMOMP. Similarly the second vector preferably comprises a plasmid vector and specifically may be pCA76kDa. Most preferably, both the first and second vectors are plasmid vectors and specifically the combination of pCAMOMP and pCA76kDa.
The two vectors are used in an immunogenic composition along with any convenient pharmaceutically-acceptable carrier. As noted above, the uses of the combination of two vectors produces an enhanced protection efficacy in comparison to the individual vectors alone. Accordingly, the first and second vectors preferably are present in the immunogenic composition in amounts such that the individual protective effect of each vector upon administration to the composition to the host is not adversely affected by the other.
The present invention, in a further aspect thereof, provides a method of immunizing a host against disease caused by infection with a strain of Chlamydia, which comprises administering to the host, which may be a human host, an effective amount of an immunogenic composition provided herein. The immunogenic composition preferably is administered intranasally.